A major goal of biomedical research is to provide protection against viral disease through immunization. One approach has been to use killed vaccines. However, large quantities of material are required for killed vaccine in order to retain sufficient antigenic mass. In addition, killed vaccines are often contaminated with undesirable products during their preparation. Heterologous live vaccines, using appropriately engineered adenovirus, which is itself a vaccine, seems like an excellent immunogen [Chanock R., JAMA, 195, 151 (1967)]. Our invention concerns vaccines using adenovirus as a vector.
Presently marketed adenovaccine comprises live, infectious adenoviruses in an enteric-coated dosage form. Upon administration to the patient to be vaccinated, the virus is carried past the upper-respiratory system (where disease-producing infection is thought to occur), and is released in the intestine. In the intestine, the virus reproduces in the gut wall, where, although it is not capable of causing adenoviral disease, nevertheless induces the formation of adenovirus antibodies, thus conferring immunity to adenoviral disease. In our invention, live, infectious adenovirus which has been engineered to contain genes coding for antigens produced by other disease-causing organisms. Upon release the virus will reproduce and separately express both the adenoviral antigen and the pathogen antigen, thereby inducing the formation of antibodies or induce cell mediated immunity to both adenovirus and the other disease-causing organism. By xe2x80x9clive virusxe2x80x9d is meant, in contradistinction to xe2x80x9ckilledxe2x80x9d virus, a virus which is, either by itself or in conjunction with additional genetic material, capable of producing identical progeny. By xe2x80x9cinfectiousxe2x80x9d is meant having the capability to deliver the viral genome into cells.
Roy, in European Patent Publication 80,806 (1983), proposed a method for producing immunity to microbial diseases by the administration of a microbe containing a foreign gene which will express an antigen of a second microbe to which immunity is conferred. He states that preferred oral preparations are enteric coated. Dubelcco proposed recombinant adenovirus vaccines in which the surface protein of adenovirus is modified to contain in its structure a segment of foreign protein which will produce a desired biological response on administration to animals. [PCT International Publication WO 83/02393 (1983)]. Davis discloses oral vaccines derived from recombinant adenoviruses. [UK Patent GB 2166349 B].
Human immunodeficiency virus type 1 (HIV-1) has been etiologically associated with acquired immunodeficiency syndrome (AIDS) and related disorders. [Barre-Sinoussi, F., Science 220: 868 (1983); Gallo, R., Science 224: 500 (1984); Popovic, M., Science 224: 497 (1984); Sarngadharan, M., Science 224: 506 (1984)]. AIDS is now a worldwide epidemic for which, currently, there is no vaccine or cure. Most of the effort for vaccine development has focused on the envelope (env) glycoprotein as an antigen which might provide protective immunity. Antisera prepared against purified gp 120 can neutralize HIV-1 in vitro. [Crowl, R., Cell 41: 979 (1985); Putney, S., Science 234: 1392 (1986); Ho, D., J. Virol. 61: 2024 (1987); Nara, P., Proc. Natl. Acad. Sci. USA 84: 3797 (1987)]. HIV-1 envelope antigen has been produced in different expression systems including Escherichia coli [Crowl, R., Cell 41: 979 (1985); Chang, T., Bio/Technology 3: 905 (1985); Dawson, G., J. Infect. Dis. 157: 149 (1988)] as well as mammalian [Chakrabarti, S., Nature 320: 535 (1986); Dewar, R., J. Virol. 63: 129 (1989); Rekosh, D., Proc. Natl. Acad. Sci. USA 85: 334 (1988); Whealy, M., J. Virol. 62: 4185 (1988)] yeast [Barr, P., Vaccine 5: 90 (1987)] and insect cells [Hu, S., Nature 328: 721 (1978); Rusche, J., Proc. Natl. Acad. Sci. USA 84: 6294 (1987)].
Live recombinant vaccinia virus expressing the entire HIV-1 env glycoprotein [Hu, S., J. Virol. 61: 3617 (1987)] or purified recombinant gp 120 env glycoprotein [Berman, P., Proc. Natl. Acad. Sci. USA 85: 5200 (1988)] were evaluated in chimpanzees as vaccine candidates. Active immunization with these vaccines induced a good cell-mediated immune response as well as cytotoxic T-cell activity to the env antigen [Zarling, J., J. Immunol. 139: 988 (1987)]. All experimental animals seroconverted as assayed by ELISA and Western blotting. However, immunized chimpanzees developed no or only low titers of neutralizing antibody to HIV-1. Challenge with live virus failed to protect chimpanzees against these vaccines. Type-specific HIV-1 neutralizing antibodies were found in chimpanzees early in infection against a variable domain (V3) within the C-terminus half of gp 120 [Goudsmit, J., Proc. Natl. Acad. Sci. USA 85: 4478 (1988)]. The recombinant gp 120 made in insect cells has also been shown to induce humoral immune response in goat (Rusche J., Proc. Natl. Acad. Sci. USA 84: 6294 (1987)]. Zagury [Nature 332: 728 (1988)] have demonstrated both anamnestic humoral and cellular immune reaction in humans using a vaccine virus recombinant expressing gp 160 [Chakrabarti, S., Nature 320: 535 (1986); Hahn, B., Proc. Natl. Acad. Sci. USA 82: 4813 (1985)]. Both group-specific cell-mediated immunity and cell-mediated cytotoxicity against infected T4 cells were also found. These results indicate that an immune state against HIV-1 can be obtained in humans using recombinant env-based vaccine. Recently, Desrosiers has shown that vaccination with inactivated whole simian immunodeficiency virus (SIV) can protect macaques against challenge with live SIV. [Proc. Natl. Acad. Sci. USA 86: 6353 (1989)]. These data provide hope that vaccine protection against human AIDS virus, HIV-1, infection may be possible.
Chanda discloses high level expression of the envelope glycoproteins of HIV-1 in the presence of rev gene using helper-independent adenovirus type 7 recombinants. [Virology 175: 535 (1990)]. Vernon discloses the ultrastructural characterization of HIV-1 gag subunit in a recombinant adenovirus vector system. [J. Gen. Virology 72: 1243 (1991)]. Vernon also discloses the preparation of the HIV-1 recombinant denoviruses Ad7-rev-gag and Ad4-rev-gag.
This invention provides a method of producing antibodies or cell mediated immunity to an infectious organism in a warm blooded mammal which comprises administering to said warm blooded mammal intranasally, intramuscularly, or subcutaneously, live recombinant adenoviruses in which the virion structural protein is unchanged from that in the native adenovirus from which the recombinant adenovirus is produced, and which contain the gene coding for the antigen corresponding to said antibodies or inducing said cell mediated immunity. The warm blooded mammal is preferably a primate, most preferably a human.
In its preferred embodiments, this invention provides a method of producing antibodies to human immunodeficiency virus (HIV-1), hepatitis B, hepatitis C, human papilloma virus, respiratory syncytial virus, rotavirus, or parainfluenza virus in a warm blooded mammal which comprises administering to said warm blooded mammal intranasally, intramuscularly, or subcutaneously, live recombinant adenoviruses in which the virion structural protein is unchanged from that in the native adenovirus from which the recombinant adenovirus is produced and which contain the gene coding for, respectively, human immunodeficiency virus, hepatitis B, hepatitis C, human papilloma virus, respiratory syncytial virus, rotavirus, or parainfluenza virus.
This invention also provides composition for producing antibodies or cell mediated immunity to an infectious organism in a warm blooded mammal, comprising live recombinant adenoviruses in which the virion structural protein is unchanged from that in the native adenovirus from which the recombinant adenovirus is produced, and which contain the gene coding for the antigen corresponding to said antibodies or inducing said cell mediated immunity, said composition being formulated in an intranasal, intramuscular, or subcutaneous dosage form.
Although this specification specifically refers to adenovirus of types 4, 5, or 7, live, infectious adenovirus of any type may be employed in this invention. Additionally, while the specification specifically refers to adenoviruses having an early region 3 (E3) deletion, adenoviruses which are attenuated, contain a temperature sensitive lesion, or a E1 deletion may also be used as a vector. Similarly, although specific reference has been made to vaccines producing antibodies to HIV, hepatitis B, hepatitis C, human papilloma virus, respiratory syncytial virus, rotavirus, or parainfluenza virus, our invention provides vaccines against any infectious agent containing an antigen to which a warm-blooded animal will produce antibodies or cell mediated immunity, and which antigen is coded for by a gene composed of up to about 3000 base pairs. Thus, for example, included within the scope of the invention are immunization against such diseases as influenza, hepatitis A, cholera, E. coli, pertussis, diphtheria, tetanus, shigellosis, gonorrhea, mycoplasma pneumonia, and the like.
In one embodiment, the method of treatment includes administering the recombinant adenovirus both prophylactically to an HIV-1 susceptible mammal and as immunotherapy following detection of HIV in said mammal. Regimens containing the following recombinant adenoviruses were used to produce the anti-HIV responses.
In a preferred embodiment, the method is a method of protecting a primate against HIV-1 infection comprising intranasal or intramuscular administration to said primate of an intranasal or intramuscular dosage of a recombinant adenovirus having a deletion in the E3 gene and an expression cassette containing a major late promoter, a tripartite leader sequence, part or all of the HIV-1 gp160 sequence and a polyadenylation signal sequence. Preferably the primate is a human. The expression cassette is inserted into the recombinant adenovirus between the E4 promoter and the inverted terminal repeat. Optionally the intranasal or intramuscular administration of recombinant adenovirus is followed by one or more intranasal or intramuscluar booster administrations of the recombinant adenovirus. The recombinant adenovirus is a serotype 4, 5 or 7 serotype adenovirus and optionally the expression cassette additionally comprises part of all of the coding sequence for the HIV-1 rev gene inserted in frame after the HIV-1 gp160 sequence and before the polyadenylation signal sequence. The HIV-1 gp160 sequence can be from the MN strain gp160 sequence or the LAV strain gp160 sequence. In an alternative embodiment, the HIV-1 gp160 sequence is replaced by a sequence encoding the gag-pro region of HV-1. In either embodiment, when the initial administration is followed by one or more intranasal or intramuscular booster administrations of the recombinant adenovirus, the last booster administration may be followed by an intramuscular injection of at least one booster immunization with an HIV-1 subunit antigen preparation, preferably containing an HIV-1 gag and/or env polypeptide sequence. For intranasal administration, the intranasal dosage administered is in the range of 1xc3x97107 pfu of virus and for intramuscular administration, the intramuscular dosage administered is in the range of 1xc3x97107 to 2xc3x97109 pfu of virus. The intranasal booster is administered in a dosage in the range of 1xc3x97107 to 1xc3x97108 pfu of virus and the intramuscular booster is administered in a dosage in the range of 1xc3x971010 to 8xc3x971010 pfu of virus. When a subunit antigen booster is employed, the subunit antigen preparation contains between 200 xcexcg and 0.5 mg of HIV-1 polypeptide.
Referring to the above table Ad4, Ad5, and Ad7 refer to human adenoviruses types 4, 5, and 7 respectively in which the E3 region has been deleted. Env refers to the HIV envelope glycoprotein (gp 160) gene. Gag refers to the HIV gag/pro gene. Rev refers to the HIV regulatory gene. Hrev refers to an altered version of the rev gene where the nucleotide sequences were changed without changing the amino acid sequence employing codons that were frequently used in human genes. The sequence of Hrev is set forth in FIG. 2. Tpl refers to the upstream adenovirus tripartite leader sequence with an intervening sequence between the first and second leaders that are positioned in front of the recombinant genes. The constructs designated Ad7-env, Ad7-gag, Ad7-gag-1, Ad4-env, Ad4-gag, Ad4-gag-1, Ad5-env, and Ad5-gag contain either the gag or the env gene from the LAV strain of HIV and the constructs Ad7-envMN, Ad4-envMN, and Ad5-envMN contain the env gene from the MN strain of HIV. The recombinant adenoviruses made from the LAV and MN strains of HIV-1 are illustrative of recombinant adenoviruses covered by this invention. This invention also covers recombinant adenoviruses which include the env and/or gag genes from other strains of HIV-1.
Both the Ad-env and Ad-envMN adenoviruses were shown to replicate in human A549 cells and expressed recombinant env antigen in vitro demonstrating their capability of generating cell mediated, humoral, and secretory immunity in a mammal.
As described in detail below, intranasal administration of Ad-HIV recombinant viruses to naive chimpanzees resulted in both priming and boosting of both humoral and cell-mediated immune responses directed at HIV recombinant antigens. The recombinant adenoviruses administered to chimpanzees were shown to produce antibodies to the env and gag proteins of HIV. IgG antibodies specific for HIV were observed in nasal, saliva, and vaginal secretions following administration of the recombinant adenoviruses and IgA antibodies specific for HIV were observed in nasal and saliva secretions. The first set of recombinant viruses (Ad7) appeared to be shed the longest period of time and induce the best anti-Ad antibody response. The results also showed that administration of Ad-HIV vaccines by the intranasal route was superior to administration of enteric-coated recombinant viruses by the oral route.
Optimum immune responses directed at HIV antigens required primary infection one booster immunization with a heterotypic recombinant Ad-HIV to elicit strong anti-HIV binding antibodies. Intranasal administration of the Ad-HIV viruses effectively primed chimpanzees to respond with high titered neutralizing antibodies to HIV-1 following subsequent HIV-1 subunit protein booster immunization.